We have isolated several novel oncogenes using an expression cloning system we have developed. These genes include ECT2, OST, TIM, NET1 and NTS1, which share common motifs for the guanine nucleotide exchange factors of the Rho family of small GTPases. The main purpose of this project is to understand the biological functions of these exchange factors at the molecular level. ECT2 catalyzes guanine nucleotide exchange on the Rho GTPases, RhoA, Rac1, and Cdc42. ECT2 is phosphorylated during G2 and M phases, and phosphorylation is required for its exchange activity. We previously showed that the ECT2 is a critical regulator of cytokinesis. In this year, we examined Rho activation during cell cycle. The level of GTP-Rho did not change significantly from S phase to prometaphase, but increased thereafter, peaking in telophase, and returned to the original level in G1 phase. The GDP-GTP exchange activity on RhoA in cell lysates increased also during mitosis with a peak in metaphase. Interestingly, expression of the dominant-negative form of ECT2 completely inhibited the rise of GTP-Rho as well as the increase of the exchange activity in telophase. These results suggest a critical role of ECT2 in Rho activation during cytokinesis. The OST protooncogene encodes a guanine nucleotide exchange factor for the Rho family of small GTPases, RhoA and Cdc42. The N-terminal domain of Ost (Ost-N) appears to negatively regulate the oncogenic activity of the protein, since deletion of this domain dramatically increases its transforming activity in NIH3T3 cells. Using a yeast two hybrid system, we identified five genes encoding proteins that can interact with Ost-N. One of them, designated OSTIP2 (Ost interacting protein 2), encoded a previously uncharacterized protein. OSTIP2 is highly expressed in skeletal muscle as a 1.2 kb transcript. Full-length OSTIP2 cDNA contained an open reading frame of 193 amino acids. Transcription-coupled translation of OSTIP2 cDNA in reticulocyte lysates revealed a protein product of 20-kDal, which corresponded to the predicted size of the protein. Bacterially-expressed glutathione S-transferase (GST)-Ostip2 fusion protein efficiently associated in vitro with baculovirus-expressed Ost. Interestingly, expression of Ostip2 in NIH3T3 cells efficiently induced foci of morphologically transformed cells. Moreover, inoculation of athymic nude mice with OSTIP2 transfectants strongly induced tumor formation. These results suggest that Ostip2 is a novel oncoprotein, which can interact with the Rho exchange factor Ost.